Abstract Cardiovascular disease remains the single greatest cause of mortality in America. In those aged 65 or older, cardiovascular disease accounts for a proportionally greater mortality rate than other age groups. Changes in cardiomyocyte Ca2+ handling, particularly a prolonged Ca2+ signal resulting in a prolonged relaxation time, can cause age-dependent cardiac dysfunction. Work showing increased left ventricular filling pressures and volumes with age provides evidence that cardiomyocytes in aged hearts bear greater longitudinal stretch than their young counterparts. My sponsor?s laboratory has novel data showing age-dependent upregulation of the stretch- activated cation channel Transient Receptor Potential Vanilloid 4 (TRPV4) in the aged myocardium. Using Young (3-4 month) and Aged (24-26 month) mice in combination with genetic (inducible cardiac-specific TRPV4 overexpression) and pharmacologic (TRPV4 inhibition) approaches, this proposal tests the central hypothesis that mechanical stretch activates TRPV4 in cardiomyocytes of the aged heart, leading to enhanced ECC and impaired diastolic relaxation. Aim 1 investigates the role of TRPV4 in stretch-induced Ca2+ homeostasis at the isolated cell level. Cardiomyocytes of Young and Aged mice will be subjected to physiologic, longitudinal stretch while measuring sarcolemmal Ca2+ entry (Mn2+ quench of fura-2), sarcoplasmic reticulum Ca2+ loading (fluo-5F, with caffeine application), spontaneous ryanodine receptor-mediated Ca2+ release events (fluo-4), and simultaneous ratiometric Ca2+ (fura-2) and force during electric field stimulation. Aim 2 extends methodology to the level of the whole organ, as isolated hearts of Young and Aged mice will be subjected to increases in preload to examine pressure development. Transgenic mice expressing the GCaMP6f Ca2+ sensor in cardiomyocytes will be utilized to examine cardiomyocyte Ca2+ dynamics in response to elevations in preload. This proposal evaluates TRPV4 as a novel target for therapeutic modulation of cardiomyocyte Ca2+ homeostasis in the aged heart. Furthermore, this proposal will give the applicant valuable training and perspective, and serve as a foundation for a future career in cardiovascular research.